News and Publications

Molecular Basis of Integrin Signaling in Hematopoietic and Vascular Cells

Integrins are signaling receptors with a twist: they transduce signals in both directions across the plasma membrane and focus extracellular cues at specific sites of adhesion between cells and the extracellular matrix. In so doing, integrins initiate changes in cell polarity, shape, cytoskeletal organization, and gene expression required for many aspects of embryonic development and for function of adult organisms. To carry out their adhesive and signaling tasks efficiently, integrins must become activated. Traditionally, activation has referred to structural changes in the receptor that are necessary for productive engagement of adhesive ligands. Recent studies suggest that this concept should be revised to include changes in the integrin that are necessary for transduction of signals into the cell. In other words, integrin signaling and activation are bidirectional and reciprocal. For example, intracellular ligands bind to integrin cytoplasmic tails in response to "inside-out" signals to regulate receptor affinity and avidity. In turn, adhesive ligands bind to the extracellular face of the integrin and initiate "outside-in" signals through activation of integrin-associated protein kinases.

One major subclass of integrins, the ß₃ integrins, includes α_IIbß₃ and α_vß₃. These receptors mediate responses of the vasculature to injury by promoting platelet adhesion and aggregation (α_IIbß₃) and endothelial cell migration and proliferation (α_vß₃). In pathologic states, ß₃ integrins promote thrombosis, neovascular proliferation, tumor angiogenesis, and metastasis. One of our goals is to understand the molecular basis of bidirectional integrin activation and signaling, with a special emphasis on the role of ß₃ integrins in blood and vascular diseases.

Stimulation of platelets by excitatory agonists increases the affinity of α_IIbß₃ for fibrinogen. Megakaryocytes have inside-out signaling similar to that of platelets, but unlike platelets, they are readily amenable to genetic manipulation. Therefore, to facilitate mechanistic studies of inside-out signaling, we established methods to generate megakaryocytes in quantity from murine embryonic stem cells. Coculture of embryonic stem cells for 8-12 days with OP9 stromal cells in the presence of thrombopoietin, IL-6, and IL-11 resulted in the development of large, polyploid megakaryocytes that produced proplatelets. These cells expressed α_IIbß₃ and platelet glycoprotein Ibα but were devoid of cell-surface markers specific for hematopoietic stem cells, erythrocytes, and leukocytes.

In response to platelet agonists, mature megakaryocytes, but not megakaryocyte progenitors, specifically bound fibrinogen through α_IIbß₃. Retrovirus-mediated expression of the gene for green fluorescent protein in these megakaryocytes did not affect cell viability or α_IIbß₃ function. On the other hand, retroviral expression of CalDAG-GEFI, a Rap1 exchange factor identified by megakaryocyte gene profiling as a candidate integrin regulator, enhanced agonist-induced activation of Rap1b and fibrinogen binding to α_IIbß₃. These results establish that embryonic stem cells are a ready source of mature megakaryocytes for integrin studies and other biological applications, and they implicate CalDAG-GEFI in inside-out signaling to α_IIbß₃.

Because CalDAG-GEFI both activates Rap1b and stimulates ligand binding to α_IIbß₃, we evaluated the role of Rap1b per se in integrin function. GFP-Rap1b chimeras were introduced into murine megakaryocytes by viral transduction. Expression of constitutively active GFP-Rap1b had no effect on unstimulated megakaryocytes, but it greatly augmented fibrinogen binding to α_IIbß₃ induced by a thrombin receptor agonist. The Rap1b effect occurred solely in cells expressing the recombinant protein and was prevented by pretreating cells with cytochalasin D or latrunculin A to inhibit actin polymerization. Rap1b-dependent fibrinogen binding to megakaryocytes was blocked by POW-2, a novel monovalent antibody Fab fragment specific for high-affinity murine α_IIbß₃. In contrast to GFP-Rap1b (V12), expression of GFP-Rap1GAP, which deactivates endogenous Rap1, inhibited agonist-induced fibrinogen binding, as did expression of dominant-negative GFP-Rap1b (N17). None of these treatments affected surface expression of α_IIbß₃. These studies establish that Rap1b can augment integrin affinity, possibly by modulating integrin interactions with the actin cytoskeleton.

Publications

Arya, M., Lopez, J.A., Romo, G.M., Cruz, M.A., Kasirer-Friede, A., Shattil, S.J., Anvari, B. Glycoprotein Ib-IX-mediated activation of integrin α_IIbß₃: effects of receptor clustering and von Willebrand factor adhesion. J. Thromb. Haemost. 1:1150, 2003.

Bertoni, A., Tadokoro, S., Eto, K., Pampori, N., Parise, L.V., White, G.C., Shattil, S.J. Relationships between Rap1b, affinity modulation of integrin α_IIbß₃, and the actin cytoskeleton J. Biol. Chem. 277:25715, 2002.

Buensuceso, C., de Virgilio, M., Shattil, S.J. Detection of integrin α_IIbß₃ clustering in living cells. J. Biol. Chem. 278:15217, 2003.

Eto, K., Leavitt, A.D., Nakano, T., Shattil, S.J. Development and analysis of megakaryocytes from murine embryonic stem cells. Methods Enzymol., in press.

Eto, K., Murphy, R., Kerrigan, S.W., Bertoni, S., Stuhlmann, H., Nakano, T., Leavitt, A.D., Shattil, S.J. Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling. Proc. Natl. Acad. Sci. U. S. A. 99:12819, 2002.

Hato, T., Ginsberg, M.H., Shattil, S.J. Integrin α_IIbß₃ and platelet aggregation. In: Platelets. Michelson, A.D. (Ed.). Academic Press, San Diego, 2002, p. 105.

Tomiyama, Y., Shiraga, M., Shattil, S.J. Platelet membrane proteins as adhesion receptors. In: Platelets in Thrombotic and Nonthrombotic Disorders: Pathophysiology, Pharmacology and Therapeutics. Gresele, P., et al. (Eds.). Cambridge University Press, New York, 2002, p. 80.

Woodside, D.G., Obergfell, A., Talapatra, A., Calderwood, D.A., Shattil, S.J., Ginsberg, M.H. The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin ß cytoplasmic domains. J. Biol. Chem. 277:39401, 2002.